- MA1-17765 - Invitrogen Antibodies CEACAM6 antibody

Submitted references Comprehensive RNA analysis of CSF reveals a role for CEACAM6 in lung cancer leptomeningeal metastases.

Li Y, Polyak D, Lamsam L, Connolly ID, Johnson E, Khoeur LK, Andersen S, Granucci M, Stanley G, Liu B, Nagpal S, Hayden Gephart M
NPJ precision oncology 2021 Oct 8;5(1):90

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of CEACAM6 was achieved by transfecting A549 with CEACAM6 specific siRNAs (Silencer® select Product # s9283, s9285). Western blot analysis (Fig. a) was performed using whole cell extracts from the CEACAM6 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with CEACAM6 Monoclonal Antibody (9A6) (Product # MA1-17765, 4 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Carcinoembryonic antigen-related cell adhesion molecule 6.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-CEACAM6 Monoclonal Antibody (9A6) (Product # MA1-17765) and a 60 kDa band corresponding to Carcinoembryonic antigen-related cell adhesion molecule 6 was observed in A549 and MCF7 but not in MIA PaCa-2 or Hep G2 which are reported to be low expressing for CEACAM6. An uncharacterized band (*) at ~58 kDa is detected across the cell lines tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), MCF7 (Lane 2), MIA PaCa-2 (Lane 3) and Hep G2 (Lane 4) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (4 µg/mL dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of CEACAM6 (Carcinoembryonic antigen-related cell adhesion molecule 6) was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with CEACAM6 Monoclonal Antibody (9A6) (Product # MA1-17765) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2500), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing lysosome like punctate structures in the cytoplasm for CEACAM6 in A549 cells. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of CEACAM6 in BOSC23 cells using a CEACAM6 Monoclonal Antibody (Product # MA1-17765) at a 1.2 µg/10^6 cells dilution, detected with a PE-conjugated secondary antibody, as shown in red. Isotype control antibody is shown in black.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 CEACAM6 mediates the migration of NSCLC cells. A A549 lung adenocarcinoma cells natively expressed high levels of CEACAM6 as opposed to H460 cells that showed undetectable levels of CEACAM6 , as measured by qPCR. A549 cells were treated for 72 h with siCEACAM6 failed to migrate toward FBS. B Western blot analysis confirmed CEACAM6 knockdown in A549 cells following treatment with siRNA. C qPCR analysis confirmed CEACAM6 knockdown in A549 cells following 72 h knockdown with siRNA. D Quantitative analysis and representative images of decreased migration of A549 cells following knockdown of CEACAM6 , as compared with siCtrl-treated cells (normalized to untreated cells). E Western blot and F qPCR confirmed elevated CEACAM6 levels in H460 cells following plasmid transfection. G Quantitative analysis and representative images showed increased cell migration following overexpression of CEACAM6, as compared with H460 cells. Student's t -test was used to evaluate the statistical significance of the difference between groups in D and G . Scale bar = 400 um in D and G images. Results are shown as the mean +- standard deviation (SD) of three independent assays.

MA1-17765

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